Polyketides are complex natural products that are produced by microorganisms such as fungi and mycelial bacteria. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including: adriamycin, epothilone, erythromycin, mevacor, rapamycin, tacrolimus, tetracycline, rapamycin, and many others. However, polyketides are made in very small amounts in microorganisms and are difficult to make or modify chemically. For this and other reasons, biosynthetic methods are preferred for production of therapeutically active polyketides. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146 and 6,410,301; Fu et al., 1994, Biochemistry 33:9321-26; McDaniel et al., 1993, Science 262: 1546-1550; Kao et al., 1994, Science, 265:509-12, and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34: 881-88, each of which is incorporated herein by reference.
The biosynthesis of polyketides may be accomplished by heterologous expression of Type I or modular polyketide synthase enzymes (PKSs). Type I PKSs are large multifunctional protein complexes, the protein components of which are encoded by multiple open reading frames (ORF) of PKS gene clusters. Each ORF of a Type I PKS gene cluster can encode one, two, or more modules of ketosynthase activity. Each module activates and incorporates a two-carbon (ketide) unit into the polyketide backbone. Each module also contains multiple ketide-modifying enzymatic activities, or domains. The number and order of modules, and the types of ketide-modifying domains within each module, determine the structure of the resulting product. Polyketide synthesis may also involve the activity of nonribosomal peptide synthetases (NRPSs) to catalyze incorporation of an amino acid-derived building block into the polyketide, as well as post-synthesis modification, or tailoring enzymes. The modification enzymes modify the polyketide by oxidation or reduction, addition of carbohydrate groups or methyl groups, or other modifications.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker regions. These regions collectively can be considered to define boundaries of the various domains. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKSs by various available methodologies. Using this method, new polyketide synthases (which produce novel polyketides) can be produced. It will be recognized from the foregoing that genetic manipulation of PKS genes and heterologous expression of PKSs can be used for the efficient production of known polyketides, and for production of novel polyketides structurally related to, but distinct from, known polyketides (see references above, and Hutchinson, 1998, Curr. Opin. Microbiol. 1:319-29; Carreras and Santi, 1998, Curr. Opin. Biotech. 9:403-11; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference).
One valuable class of polyketides includes the leptomycins and their analogs (FIG. 1). These compounds are selective inhibitors of protein export from the cell nucleus and thus affect the cellular location of proteins. The function of many key proteins and transcription factors involved in cell growth can be regulated by their cellular location. For instance, the tumor suppressor p53 normally resides in the cell nucleus where its activation promotes cell-cycle arrest and apoptotic cell death. Mislocation of p53 into the cytoplasm, especially its dominant negative mutant forms, is associated with development of many types of cancer. Nuclear factor kB (NFkB) is a transcriptional activator that targets genes involved in cell proliferation and apoptosis. It is constitutively activated in certain cancer cells, aiding tumor resistance to radiation and cancer chemotherapy drugs. NFkB resides in the cytoplasm in an inactive form complexed with the inhibitor of nuclear factor IkB; upon stimulation by factors such as TNF-a or CD-40 ligand, events are set in place that remove IkB and allow importation of NFkB into the cell nucleus.
Leptomycin B (LMB; also known as CI-940 or elactocin) and the ratjadones (FIG. 2) are the only known low molecular weight inhibitor of nuclear transport. Because of the structural similarities, the kazusamycins, leptofuranins and callystatins are also implicated. Callystatins come from a marine sponge whereas all the other compounds are bacterial metabolites. All of these molecules are exceptionally potent, typically displaying IC50 values in the 100 picomolar to 10 nanomolar range.
Protein export from the cell nucleus requires a nuclear export signal (NES) as a domain in the exported protein, CRM1 (exportin-1) to recognize the NES and Ran, a Ras-like GTPase. In the nucleus CRM1 forms a complex with the NES-protein and Ran/GTP, then the complex is translocated through the nuclear pore complex into the cytoplasm. There, the Ran GTPase activating protein (RanGAP), found only in the cytoplasm, promotes hydrolysis of Ran/GTP to Ran/GDP, causing release of the NES-protein.
The high potency and novel mechanism of action prompted an investigation of the antitumor activity of LMB in mouse murine and xenograph cancer models. Activity was observed at low doses against adriamycin, amsacrine and mitoxantrone resistant P388 leukemia, other leukemias, B16 melanoma, Ridgway osteogenic and M5076 sarcomas and mammary adenocarinoma. Acute toxicity appeared to be gastrointestinal and was exacerbated upon more frequent or oral administration of the drug. The maximum tolerated dose (MTD) in mice ranged from 0.12 to 1 mg/kg, as a function of dosing schedule.
LMB has also attracted considerable interest as a biochemical tool to study the role and regulation of nucleo-cytoplasmic shuttling proteins and for its potential therapeutic use in combination with other drugs. Vigneri and Wang, “Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase,” Nature Medicine (2001) 7:228-234, describes combined treatment of cultured CML cells with STI-571 and LMB. STI-571 effectively masks the ability of Bcr-Abl to be retained preferentially in the cytoplasm; upon nuclear importation of the drug-inactivated protein, LMB inhibits nuclear export of Bcr-Abl and withdrawal of STI-571 releases the ability of the constitutively activated Abl component to induce apoptosis. While the effect of either drug alone is fully reversible (STI-571 does not permanently inhibit Bcr-Abl and nuclear export is restored by synthesis of fresh CRM1), their combined use caused irreversible and complete killing of the Bcr-Abl transformed cells. Such treatment also preferentially eliminated mouse bone marrow cells that express Bcr-Abl. This strategy can overcome the main limitation of acute CML treatment with STI-571, which is acquired drug resistance due to mutation or overexpression of Bcr-Abl.
LMB has other types of potential therapeutic uses. Because it can promote nuclear retention of the p53 tumor suppressor protein, treatment with LMB can lead to p53 activation in the nucleus, which results in cell-cycle arrest and apoptosis. Combined use LMB and actinomycin D can reactivate p53 and prevent its degradation by HPV E6 protein in cervical carcinoma cells infected with human papillomavirus. LMB can also potentiate the effect of rapamycin, an emerging cancer drug, by blocking nuclear export of mTOR, the protein kinase target of rapamycin that controls the activity of two transcription factors. The antiviral activity of LMB has been elucidated as resulting from inhibition of the nuclear export of the HIV-1 Rev protein and Rev-dependent unspliced and partially spliced mRNA, which is an early step in viral replication. LMB interferes with cyclinB1/Cdc2, cyclinD1/CDK4 and TGF-beta dependent signaling also, suggesting possible uses against cancers with aberrant signaling involving these actors. A synthetic HIV-1 Rev inhibitor, PKF050-638 (FIG. 2), has been developed that mimics the activity of LMB.
Two limitations have to be overcome to increase the potential for development of LMB into an effective anticancer or antiviral drug. One, a reliable source of pure drug must be developed, because “The use of LMB . . . has been hampered by the variability of the quality of LMB production lots” (D. Daelemans et al. 2002, “A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export” Proc. Natl. Acad. Sci. USA 99: 14440-5). This is not surprising given the close structural similarity of leptomycin-like compounds isolated from their natural sources (FIG. 1). In fact, at least 5 different forms of leptomycins have been detected in the culture extracts of the ATCC 39366 strain and 6 forms in another LMB producer. Two, a less toxic form of LMB would be more appealing for drug development studies. Even though the drug's effects have been reported to be fully reversible, toxicity is likely to be mechanism-related and exhibited in different bodily tissues given the widespread role of CRM1-mediated protein export. The available SAR data (FIG. 2) are insufficient for designing a less toxic analog. Analog production and evaluation will require both chemical and microbiological approaches, because little effort towards the total synthesis of LMB has been reported.
The following data suggest that analogs with an acceptable therapeutic index could be found. LMB displayed an approx. 250-fold difference in activity between a Rev-dependent assay and cytotoxicity to the same cells in vitro and PKF050-638 had a 75-fold difference in the same two assays (FIG. 2). These data show that LMB itself can have a good therapeutic window in certain instances. It is thus likely that less toxic LMB analogs can be discovered as a consequence of differential binding to CRM1 or pharmacokinetic behavior that modulates their distribution, half-life or metabolism.
Given the promise of leptomycin B in the treatment of conditions and diseases characterized by undesired cellular hyperproliferation, there thus exists an unmet need for a production system that can provide large quantities of leptomycin B in a form substantially free of minor congeners and other impurities. The present invention meets this need by providing the biosynthetic genes responsible for the production of leptomycins and providing for their expression in heterologous hosts. Further, there is an unmet need for analogs of leptomycins potentially useful in the treatment of viral diseases. The present invention meets this need by providing the means for biological generation of leptomycin analogs through genetic engineering of the biosynthetic genes.